27.23 Pyrimidines and Purines

Pyrimidines and Purines In order to understand the structure and properties of DNA and RNA, we need to look at their structural components. We begin with certain heterocyclic aromatic compounds called pyrimidines and purines.

Pyrimidines and Purines Pyrimidine and purine are the names of the parent compounds of two types of nitrogen- containing heterocyclic aromatic compounds. N N N N N NH PyrimidinePurine

Important Pyrimidines Pyrimidines that occur in DNA are cytosine and thymine. Cytosine and uracil are the pyrimidines in RNA. HNHNHNHN NHNHNHNHOO Uracil HNHNHNHN NHNHNHNHOO CH 3 Thymine HNHNHNHN NHNHNHNH NH2NH2NH2NH2 O Cytosine

Important Purines Adenine and guanine are the principal purines of both DNA and RNA. Adenine N N NH2NH2NH2NH2N NHNHNHNH GuanineO HNHNHNHN NHNHNHNH N N H2NH2NH2NH2N

Caffeine and Theobromine Caffeine (coffee) and theobromine (coffee and tea) are naturally occurring purines. Caffeine N NON N H3CH3CH3CH3C O CH 3 TheobromineO HNHNHNHN N N N O

27.24 Nucleosides

Nucleosides The classical structural definition is that a nucleoside is a pyrimidine or purine N-glycoside of D -ribofuranose or 2-deoxy- D -ribofuranose. Informal use has extended this definition to apply to purine or pyrimidine N-glycosides of almost any carbohydrate. The purine or pyrimidine part of a nucleoside is referred to as a purine or pyrimidine base.

Uridine and Adenosine Uridine and adenosine are pyrimidine and purine nucleosides respectively of D -ribofuranose. Uridine (a pyrimidine nucleoside) Adenosine (a purine nucleoside) ON HOCH 2 HNHNHNHN O OHHO O N N N N O OHHO NH2NH2NH2NH2

27.25 Nucleotides

Nucleotides Nucleotides are phosphoric acid esters of nucleosides.

Adenosine 5'-Monophosphate (AMP) Adenosine 5'-monophosphate (AMP) is also called 5'- adenylic acid. N N N N O OHHO NH2NH2NH2NH2 OCH 2 P HO O HO

Adenosine 5'-Monophosphate (AMP) Adenosine 5'-monophosphate (AMP) is also called 5'- adenylic acid. N N N N O OHHO NH2NH2NH2NH2 OCH 2 P HO O HO 1' 2'3' 4' 5'

Adenosine Diphosphate (ADP) N N N N O OHHO NH2NH2NH2NH2 OCH 2 P O O HO P O HO HO

Adenosine Triphosphate (ATP) N N N N O OHHO NH2NH2NH2NH2 OCH 2 P O O HO P O HO O P O HO HO

ATP Stores Energy AMP ADP ATP Each step is endothermic. Energy for each step comes from carbohydrate metabolism (glycolysis). Reverse process is exothermic and is the source of biological energy.  G° for hydrolysis of ATP to ADP is –35 kJ/mol

Adenosine 3'-5'-Cyclic Monophosphate (cAMP) Cyclic AMP is an important regulator of many biological processes. N N N N O OHO NH2NH2NH2NH2 CH 2 P O HO O

27.26 Nucleic Acids

Nucleic Acids Nucleic acids are polymeric nucleotides (polynucleotides). 5' Oxygen of one nucleotide is linked to the 3' oxygen of another.

Fig A section of a polynucleotide chain.

27.27 Structure and Replication of DNA: The Double Helix

Composition of DNA Erwin Chargaff (Columbia Univ.) studied DNAs from various sources and analyzed the distribution of purines and pyrimidines in them. The distribution of the bases adenine (A), guanine (G), thymine (T), and cytosine (C) varied among species. But the total purines (A and G) and the total pyrimidines (T and C) were always equal. Moreover: %A = %T, and %G = %C

Composition of Human DNA Adenine (A) 30.3%Thymine (T) 30.3% Guanine (G) 19.5%Cytosine (C) 19.9% Total purines: 49.8%Total pyrimidines: 50.1% For example: PurinePyrimidine

Base Pairing Watson and Crick proposed that A and T were equal because of complementary hydrogen bonding. 2-deoxyribose 2-deoxyribose AT

Base Pairing Likewise, the amounts of G and C were equal because of complementary hydrogen bonding. 2-deoxyribose 2-deoxyribose GC

The DNA Duplex Watson and Crick proposed a double-stranded structure for DNA in which a purine or pyrimidine base in one chain is hydrogen bonded to its complement in the other.

Fig Two antiparallel strands of DNA are paired by hydrogen bonds between purine and pyrimidine bases.

Fig Helical structure of DNA. The purine and pyrimidine bases are on the inside, sugars and phosphates on the outside.

Fig DNA Replication C G T A As the double helix unwinds, each strand acts as a template upon which its complement is constructed.

Fig DNA Replication A C G T T' A' G' C'

27.28 DNA-Directed Protein Biosynthesis

DNA and Protein Biosynthesis According to Crick, the "central dogma" of molecular biology is: "DNA makes RNA makes protein." Three kinds of RNA are involved. messenger RNA (mRNA) transfer RNA (tRNA) ribosomal RNA (rRNA) There are two main stages. transcription translation

Transcription Transcription is the formation of a strand of mRNA using one of the DNA strands as a template. The nucleotide sequence of the mRNA is complementary to the nucleotide sequence of the DNA template. Transcription begins at the 5' end of DNA and is catalyzed by the enzyme RNA polymerase.

Transcription AGGTCACTG TCCAGTGAC A T G C T T T T T T C C C C C A A A A A G G A G G G As double-stranded DNA unwinds, a complementary strand of mRNA forms at the 5' end. 5' 3'

Transcription AGGTCACTG TCCAGTGAC A T G C T T T T T T C C C C C A A A A A G G A G G G A A G G U C 5' 3' Uracil is incorporated in RNA instead of thymine.

Translation The nucleotide sequence of mRNA codes for the different amino acids found in proteins. There are three nucleotides per codon. There are 64 possible combinations of A, U, G, and C. The genetic code is redundant. Some proteins are coded for by more than one codon.

Table 27.4: mRNA Codons AlanineArginineAsparagineAspartic AcidCysteine GCUGCACGUCGA AAUGAU UGU GCCGCGAGACGC AACGACUGC CGGAGG Glutamic acidGlutamineGlycineHistidineIsoleucine GAACAAGGUGGACAUAUUAUA GAGCAGGGCGGGCACAUC LeucineLysineMethioninePhenylalanineProline UUACUUAAAAUGUUUCCUCCA CUAUUGAAGUUCCCC CG CUCCUG SerineThreonineTryptophanTyrosineValine UCUUCA ACUACAUGGUAUGUUGUA AGUUCC ACCACGUACGUC GUG UCGAGC

Transfer tRNA There are 20 different tRNAs, one for each amino acid. Each tRNA is single stranded with a CCA triplet at its 3' end. A particular amino acid is attached to the tRNA by an ester linkage involving the carboxyl group of the amino acid and the 3' oxygen of the tRNA.

Phenylalanine tRNA

OCCHCH 2 C 6 H 5 NH 3 +O A C C A A A A A A A A A A A A A A A A A C C C C C C C C CC C C U U U U U U U U U U U G G G GG G G G G G G G U GG G G C This AAA triplet is complementary to a UUU triplet of mRNA; it is an anticodon.

27.29 DNA Sequencing

DNA Sequencing Restriction enzymes cleave the polynucleotide to smaller fragments. These smaller fragments ( base pairs) are sequenced. The two strands are separated.

DNA Sequencing Single stranded DNA divided in four portions. Each tube contains adenosine, thymidine, guanosine, and cytidine plus the triphosphates of their 2'-deoxy analogs. POCH 2 OHO O OOHP O OH P O HO base H HOHOHOHO O

DNA Sequencing The first tube also contains the 2,'3'-dideoxy analog of adenosine triphosphate (ddATP); the second tube the 2,'3'-dideoxy analog of thymidine triphosphate (ddTTP), the third contains ddGTP, and the fourth ddCTP. POCH 2 OHO O OOHP O OH P O HO base HH O

DNA Sequencing Each tube also contains a "primer," a short section of the complementary DNA strand, labeled with radioactive phosphorus ( 32 P). DNA synthesis takes place, producing a complementary strand of the DNA strand used as a template. DNA synthesis stops when a dideoxynucleotide is incorporated into the growing chain.

DNA Sequencing The contents of each tube are separated by electrophoresis and analyzed by autoradiography. There are four lanes on the electrophoresis gel. Each DNA fragment will be one nucleotide longer than the previous one.

Figure T TGTGTGTG TGATGATGATGA TGACTGACTGACTGAC TGACATGACATGACATGACA TGACATTGACATTGACATTGACAT TGACATATGACATATGACATATGACATA TGACATACTGACATACTGACATACTGACATAC TGACATACGTGACATACGTGACATACGTGACATACG TGACATACGTTGACATACGTTGACATACGTTGACATACGT ddAddTddGddC Sequence of fragment

Figure T TGTGTGTG TGATGATGATGA TGACTGACTGACTGAC TGACATGACATGACATGACA TGACATTGACATTGACATTGACAT TGACATATGACATATGACATATGACATA TGACATACTGACATACTGACATACTGACATAC TGACATACGTGACATACGTGACATACGTGACATACG TGACATACGTTGACATACGTTGACATACGTTGACATACGT ddAddTddGddC A ACACACAC ACTACTACTACT ACTGACTGACTGACTG ACTGTACTGTACTGTACTGT ACTGTAACTGTAACTGTAACTGTA ACTGTATACTGTATACTGTATACTGTAT ACTGTATGACTGTATGACTGTATGACTGTATG ACTGTATGCACTGTATGCACTGTATGCACTGTATGC ACTGTATGCAACTGTATGCAACTGTATGCAACTGTATGCA Sequence of fragment Sequence of original DNA